We have previously demonstrated that human tumor infiltrating lymphocytes (TIL) can specifically recognize tumor associated antigens (Ag). This can occur in a MHC class I or class II context (mediated by CD8+ or CD4+ T cells, respectively). Earlier efforts have concentrated on CD8+ cells infiltrating melanoma lesions, which can react with tumor associated Ag present on autologous and select allogeneic melanomas, as well as some Ewing's sarcomas (another tumor of neural crest origin). Efforts are underway in different sections of the Surgery Branch to directly identify the relevant CD8 tumor Ag, using peptide isolation from tumor cells as well as gene cloning techniques. We have chosen to focus on CD4+ T cell responses to melanoma, about which very little is known to date. Recruitment of CD4+ immune activity may be crucial to the success of immune strategies for cancer therapy. 1. Documenting CD4+ T cell responses to melanoma. We have established model systems using autologous T cells and cultured tumor cell lines. Many cultured melanomas fail to express MHC class II molecules spontaneously. Using IFN-gamma IFN-gamma to enhance expression of class II molecules, we have shown that class II-restricted CD4+ melanoma TIL can secrete the cytokines IFN-gamma, GM-CSF, and/or TNF-alpha in response to autologous tumor cells. This has been observed in five of seven patients studied thus far. 2. Developing bioassays to screen allogeneic tumors for shared Ag. Because of the extreme molecular diversity of human class II molecules, we have developed a system for screening allogeneic tissues for the presence of shared tumor-associated Ag, using autologous EBV-transformed B cells as Ag presenting cells. Reagents have been developed from three melanoma patients thus far. To date, only one patient's CD4+ TIL have recognized an Ag shared on allogeneic melanomas. This Ag does not seem to be present on normal tissues. 3. Identifying CD4 recognized melanoma Ag. Studies are in progress to isolate tumor peptides recognized by CD4+ melanoma TIL, using immunoaffinity chromatography to isolate class II-peptide complexes from melanoma cells. In addition, another system for tumor Ag isolation is being developed, using gel chromatography to separate individual tumor- derived proteins for processing and presentation by EBV-B cells.